The present invention relates to the Plk protein having mutation(s) and the gene encoding said protein.
Polo gene which acts during mitosis in a Drosophila cell has been reported. As a human gene similar to the Polo gene, Plk gene is known which comprises 2137 nucleotides (whole cDNA sequence) including a coding region consisting of 1809 nucleotides and encodes a protein of 603 amino acids (Golsteyn, R. M., et al., J. Cell Sci., 107, 1509-1517, 1994; Hamanaka, R., et al., Cell Growth Differ., 5, 249-257, 1994). When the function of protein is inhibited by antibody, abnormal cell division is observed (Lane, H. A. and Nigg, E. A., J.Cell Biol., 135, 1701-1713, 1996).
The Plk gene is a phosphorylation enzyme having a kinase domain at its N-terminus. The gene also has a domain called polo-box in its middle, which has been revealed to participate in localization of the protein (Lee, K. S., et al., Proc. Natl. Acad. Sci. USA, 96, 14360-14365, 1999). Furthermore, it has been shown that a proteasome is involved in degradation of the Plk protein (Ferris., D. K., et al., Biochem. Biophys. Res. Commun., 252, 340-344, 1998); and that the Plk protein is associated with tubulin which is a cytoskeleton (Feng, Y., et al., Biochem. J., 339, 435-442, 1999). Thus, Plk is considered to be subjected to various regulation and to control the progress of cell cycles. However, physiological roles, functions and the like of the Plk gene remain unknown at present, and elucidation of these features has been desired in the fields of basic scientific researches, biology, pharmaceuticals and the like.
The inventors of the present invention have conducted various studies on physiological functions of the Plk protein, and found that Hsp90 protein, a molecular chaperone which interacts with various protein molecules, was involved in stabilization of Plk protein; and that Hsp90 protein binds to the C-terminal domain of the Plk protein so as to stabilize the protein. The inventors conducted further studies and found that the Plk protein having mutation(s) at the C-terminal domain was present in malignant tumor cells. They also found that affinity of the mutant protein with Hsp90 decreases, which resulted in lack of stabilization of the Plk protein. The present invention was achieved on the basis of these findings.
The present invention thus provides the mutant Plk protein having mutation(s) in the C-terminal domain specified by amino acid residues of from 439 to 603 of the amino acid sequence of the Plk protein, wherein the mutation(s) decreases affinity with Hsp90; and the gene encoding said mutant Plk protein. The aforementioned mutant Plk protein is not stabilized by Hsp90 protein in the cells. For this reason, cells having the gene encoding said mutant Plk protein can not supply a physiologically required amount of Plk protein, which causes abnormalities in the cell such as canceration or the like. According to one embodiment, the present invention provides the aforementioned mutant Plk protein or the aforementioned Plk gene which is involved in formation of malignant tumors.
The present invention further provides:
a method for detecting abnormal cells which comprises the a step of detecting the mutant Plk protein having mutation(s) in the C-terminal domain specified by amino acid residues of from 439 to 603 of the amino acid sequence of the Plk protein wherein the mutation decreases in affinity with Hsp90, or the gene encoding said mutant Plk protein;
use of the mutant Plk protein for detecting abnormal cells, wherein said mutant Plk protein has mutation(s) in the C-terminal domain specified by amino acid residues of from 439 to 603 of the amino acid sequence of the Plk protein and wherein the mutation decreases affinity with Hsp90 protein, or the gene encoding said mutant Plk protein; and
use of the mutant Plk protein as a tumor marker, wherein said mutant Plk protein has mutation(s) in the C-terminal domain specified by amino acid residues of from 439 to 603 of the amino acid sequence of the Plk protein, and wherein the mutation decreases affinity with Hsp90 protein, or the gene encoding said mutant Plk protein. An example of the abnormal cells includes malignant tumor cells, and detection of said abnormal cells enables diagnosis of malignant tumors. An embodiment of the use of the aforementioned Plk gene includes detection of the gene capable of interacting with said gene.
Furthermore, the present invention provides the above mutant Plk protein or a homologue thereof which can function in mammalian animals as well as in humans. The invention further provides a method for creating abnormal cells which comprises the step of introducing the gene into cells which encodes the mutant Plk protein having mutation(s) in the C-terminal domain specified by amino acid residues of from 439 to 603 of the amino acid sequence of the Plk protein wherein the mutation decreases affinity with Hsp90; and abnormal cells created by the above method. An example of the abnormal cell includes malignant tumor cells, and creation of these abnormal cells enables establishment of a system for screening antitumor drugs.
From another point of view, the invention provides antibody which specifically recognizes the mutant Plk protein having mutation(s) in the C-terminal domain specified by amino acid residues of from 439 to 603 of the amino acid sequence of the Plk protein wherein the mutation decreases affinity with Hsp90 protein, or specifically recognizes the gene encoding said mutant Plk protein. An example of the antibody preferably include a monoclonal antibody, and the use of this antibody enables detection of malignant tumor cell(s) having the above mutant Plk protein or mutant Plk gene.
By using the mutant Plk protein or the mutant Plk gene provided by the present invention, abnormal cell(s) such as malignant tumor cells can be readily detected, which enables, for example, genetic diagnosis of malignant tumors. Furthermore, the mutant Plk protein is useful for pathologic elucidation, diagnosis, and treatment of malignant tumors or apoptosis-related diseases.